Terminal respiration in heart muscle as well as in other tissues and in the cells of all eucaryotic organisms occurs in a mitochondrial membrane containing as many as five respiratory complexes. These complexes, like many other enzymes associated with membrane function, are composed of a number of subunit proteins and act as energy transducers that catalyze not only respiratory processes including oxidative phosphorylation but also vital membrane functions such as transport of ions and substrates. Typical of these complexes is Complex III which catalyzes the reduction of cytochrome c by ubiquinol and contains possibly 10 or more subunit proteins. Little is known about the properties, structural interrelationships and function of at least six of these subunits in context of the respiratory and membrane function of the complex. Recently, we have isolated a protein fraction from purified Complex III that appears to consist of low-molecular weight proteins (less than 10,000 daltons) having the solubility properties of proteolipids. Although similar proteins have been isolated from other membrane complexes their function has remained obscure. We have also demonstrated that the measurement of accessibility of the subunits of Complex III to alkylation by radiolabeled protein modifiers (differential labeling) can detect changes in structural and functional interrelationships among the subunits of Complex III. We therefore propose to characterize further with respect to properties and function the proteolipids isolated from Complex III and to utilize the technique of differential labeling to better define the contributions of the individual subunits of Complex III with respect to the overall quaternary structure and the well-defined functional properties of the complex. To facilitate these studies the use of high performance liquid chromatography (HPLC) in the separation of the subunit proteins of Complex III will be investigated; in addition, the effects of several antimycin like inhibitors on the properties of Complex III as listed above will be examined.